The objective is to define the role of diadenosine 5 feet,5''',p1,p4-tetraphosphate (Ap4A) in the priming of DNA synthesis in an in vitro eukaryotic system. This novel dinucleotide has been found in a wide variety of living cells, increases in concentration 50-100 fold before cell division, binds to subunit of DNA polymerase Alpha, and stimulates DNA synthesis in an in vitro system containing highly purified DNA polymerase Alpha from HeLa cells, a synthetic double stranded octadecamer deoxynucleotide 5 feet-d(GGAGGCTTTTTTGGAGGC). (CCTCCGAAAAAACCTCCG)d-5 feet as a template, whose primary sequence is taken from the origin region of DNA replication of SV40, and Ap4A as the primer. A nascent DNA chain is formed with covalent attachment to Ap4A. We wish to explore what primary structural characteristics of this template are necessary for recognition by the enzyme, why only dA residues are at present added to the incipient chain, and whether structural variants of Ap4A will bind to the subunit of DNA polymerase Alpha and either function as chain primers or as inhibitors.